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Bradford Protein Assay

Protein Concentration Determination by the Bradford Assay

A simple procedure for the determination of protein concentration in solutions is the Bradford protein assay which was described first by Bradford (Bradford et al., 1976).

An estimation of protein concentration is essential to be done rapidly and accurately in many fields of protein study. The Bradford assay has become the preferred method for quantifying protein in many laboratories. This technique is simpler, faster, and more sensitive than the Lowry method. Furthermore, when compared with the Lowry method, it is subject to less interference by common reagents and nonprotein components of biological samples.

The Bradford assay relies on the binding of the dye Coomassie Blue G-250 to protein.

Detailed studies indicate that the free dye can exist in four different ionic forms for which the pKa values are 1.15, 1.82, and 12.4. Of the three charged forms of the dye that predominate in the acidic assay reagent solution, the more cationic red and green forms have absorbance maxima at 470 nm and 650 nm, respectively. In contrast, the more anionic blue form of the dye, which binds to protein, has an absorbance maximum at 590 nm.

Thus, the quantity of protein can be estimated by determining the amount of dye in the blue ionic form. This is usually achieved by measuring the absorbance of the solution at 595 nm.

The dye appears to bind most readily to arginyl and lysyl residues of proteins. This specificity can lead to variation in the response of the assay to different proteins, which is the main drawback of the method. The original Bradford assay shows large variation in response between different proteins. Several modifications to the method have been developed to overcome this problem.

However, these changes generally result in a less robust assay that is often more susceptible to interference by other chemicals. Consequently, the original method devised by Bradford remains the most convenient and widely used formulation. Two types of assay are described here: the standard assay, which is suitable for measuring between 10 and 100 µg of protein, and the microassay, which detects between 1 and 10 µg of protein. The latter, although more sensitive, is also more prone to interference from other compounds because of the greater amount of sample relative to dye reagent in this form of the assay.